Photosynthesis is inhibited by high temperatures that plants are likely to experience under natural conditions. Both increased thylakoid membrane ionic conductance and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) deactivation have been suggested as the primary cause. The moderately heat-tolerant crop Pima S-6 cotton (Gossypium barbadense) was used to examine heat stress-induced inhibition of photosynthesis. Previous field-work indicated that moderate heat stress (T = 35–45 °C) is associated with very rapid leaf temperature changes. Therefore, a system was devised for rapidly heating intact, attached leaves to mimic natural field heat-stress conditions and monitored Rubisco activation, carbon-cycle metabolites, thylakoid ionic conductance, and photosystem I activity. As a proxy for NADPH and stromal redox status the activation state of NADP-malate dehydrogenase (NADP-MDH) was measured. In dark-adapted cotton leaves, heating caused an increase in thylakoid permeability at temperatures as low as 36 °C. The increased permeability did not cause a decline in adenosine 5′-triphosphate (ATP) levels during steady-state or transient heating. Rapid heating caused a transient decline in ribulose 1,5-bisphosphate without a decrease in Rubisco activation. Sustained heating caused a decline in Rubisco activation and also oxidized the stroma as judged by NADP-MDH activation and this is hypothesized to result from increased cyclic photophosphorylation, explaining the maintenance of ATP content in the face of increased thylakoid membrane ion leakiness.